Targeted Analysis of Mitochondrial Protein Conformations and Interactions by Endogenous ROS-Triggered Cross-Linker
Wen Zhou1,2, Yuwan Chen1,2, Wenxin Fu1,3
1State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, China.
This study introduces novel nanoparticles that target mitochondria to analyze protein conformations and interactions in living cells. This method enhances understanding of mitochondrial protein dynamics and biological functions without genetic modification.
Area of Science:
- Biochemistry
- Cell Biology
- Nanotechnology
Background:
- Mitochondrial protein conformation and interaction studies are vital for understanding cellular function.
- Existing chemical cross-linking mass spectrometry (CX-MS) methods face limitations in analyzing mitochondrial proteins in unmodified cells.
- In situ analysis of mitochondrial protein dynamics is challenging.
Purpose of the Study:
- To develop a novel method for in situ analysis of mitochondrial protein conformations and interactions.
- To overcome the limitations of current CX-MS techniques for mitochondrial proteomics.
- To enable deep analysis of mitochondrial proteins in living cells without genetic manipulation.
Main Methods:
- Development of reactive oxygen species (ROS)-responsive cross-linker delivery nanoparticles (R-CDNP).
- R-CDNP incorporates mitochondria-targeting (triphenylphosphine), ROS-responsive (thioketal), loading (PLGA), and cross-linker (DSS) modules.
- Utilized R-CDNP for in situ cross-linking and mass spectrometry analysis in HepG2 cells.
Main Results:
- Successfully identified 2103 cross-linked sites across 572 mitochondrial proteins.
- Revealed 1718 intra-links, indicating dynamic protein conformations, including chaperone ATP-dependent cycles.
- Discovered 385 inter-links, highlighting dynamic interactions within OXPHOS complexes and identifying 27 potential novel interactions.
Conclusions:
- R-CDNP enables dynamic conformation and interaction analysis of mitochondrial proteins in living cells.
- This approach significantly advances the understanding of mitochondrial protein functions.
- The R-CDNP platform offers a powerful tool for mitochondrial research and disease mechanism studies.
More Related Videos
08:33Author Spotlight: An Optimized Automated Method for Investigating Retinoic Acid Receptors in Neuronal Mitochondria
Published on: July 28, 2023
08:37Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution
Published on: June 1, 2017
Related Concept Videos
01:19Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
01:39Mitochondrial Protein Sorting
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...